SF3B1 c.1874G>A, p.Arg625His

Reasoning

SF3B1 p.Arg625His lies at residue R625, which the Cancer Hotspots source identifies as a statistically significant hotspot. PMID:23861464 independently reports recurrent R625 substitutions (R625H, R625C, R625P, and R625L) in uveal melanoma. The variant is ultra-rare in gnomAD v4.1 (1/1,614,012 alleles; total AF 6.20e-7), providing no evidence of common benign variation. The hotspot extraction is flagged as partial, so the source display should receive human confirmation.

Rule

Located in a mutational hot spot and/or critical and well-established functional domain (e.g. active site of an enzyme) without benign variation.

Evidence considered

• Cancer Hotspots reports that SF3B1 R625 lies in a statistically significant hotspot; the record is labeled as partially parsed.
• PMID:23861464 reports p.R625H, p.R625C, p.R625P, and p.R625L in uveal melanoma tumors and describes SF3B1 mutations as recurrent cancer-associated mutations.
• gnomAD v4.1 reports the queried allele once among 1,614,012 alleles (total AF 6.195740799944486e-7; no homozygotes).

Still missing

• The Cancer Hotspots record was only partially parsed and should be visually confirmed.

Sources

Reasoning

PM2 is met at supporting strength under the supplied generic threshold. The variant is absent from gnomAD v2.1 and present only once in gnomAD v4.1, with total AF 6.195740799944486e-07 (1/1614012 alleles), no homozygotes, and AF below the PM2 supporting cutoff of 0.0001. No VCEP-specific population source governs this case, so the all-comers gnomAD totals are the default; the available record contains no non-cancer subset requiring substitution.

Rule

Absent from controls (or at extremely low frequency if recessive) in Exome Sequencing Project, 1000 Genomes or Exome Aggregation Consortium.

Evidence considered

• gnomAD v2.1: variant absent.
• gnomAD v4.1: total AF=6.195740799944486e-07, AC=1, AN=1614012, homozygotes=0; the highest observed subpopulation point estimate is Finnish AF=1.5624023498531342e-05 from 1/64004 alleles, still below 0.0001, although it is not used as the filtering metric for BA1/BS1.
• Published generic PM2 supporting threshold supplied for this case: <=0.0001, SVI recommendation to downgrade PM2 to Supporting (ClinGen SVI 2020; PMID:25741868).

Sources

Reasoning

The variant is a missense substitution, SF3B1 NM_012433.4:c.1874G>A (p.Arg625His), so the applicable generic PP3/BP4 path is REVEL only; SpliceAI and BayesDel are not used for this missense assessment. REVEL is 0.875, which meets the supplied generic PP3 moderate threshold of >= 0.773 from the ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997). The ACMG/AMP caveat that multiple computational predictions are one evidence source is respected; no additional predictor is double-counted.

Rule

Multiple lines of computational evidence support a deleterious effect on the gene or gene product (conservation, evolutionary, splicing impact, etc.). ... Missense variant -> REVEL only. ... PP3 (moderate): >= 0.773 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Local REVEL predictor lookup: SF3B1 NM_012433.4:c.1874G>A (p.Arg625His), REVEL score 0.875.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997): generic PP3 moderate threshold >= 0.773; the observed score meets it.
• Richards et al. 2015 ACMG/AMP guidance (PMID:25741868), Computational (In Silico) Predictive Programs: different in-silico tools are treated as a single piece of evidence rather than independent evidence; no double-counting was applied.

Sources

Reasoning

ClinVar contains an exact-match Likely pathogenic classification for NM_012433.4(SF3B1):c.1874G>A (p.Arg625His), but its review status is criteria provided, single submitter, with zero expert-panel submissions. Under the required generic rule, a non-expert laboratory submission cannot trigger PP5.

Rule

Reputable source recently reports variant as pathogenic, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 376534, exact variant NM_012433.4(SF3B1):c.1874G>A (p.Arg625His): Likely pathogenic, criteria provided, single submitter; one submission and zero expert-panel submissions.
• The ClinVar record is an ordinary laboratory submission, so its classification is not used to trigger PP5.

Still missing

• No exact-variant ClinVar expert-panel Pathogenic or Likely pathogenic classification is available.

Sources

Reasoning

The variant is a missense substitution, so BP4 must be assessed through the REVEL-only path; the SpliceAI max delta of 0.049 is not used for a missense BP4 determination, and BayesDel has no generic ACMG-strength calibration. REVEL is 0.875, above the supplied generic BP4 supporting threshold of <= 0.29 and not within any BP4-calibrated benign range. Therefore BP4 is not met; the applicable REVEL result does not support a benign computational effect.

Rule

Multiple lines of computational evidence suggest no impact on gene or gene product (conservation, evolutionary, splicing impact, etc). ... Missense variant -> REVEL only. ... BP4 (supporting): <= 0.29 — ClinGen SVI REVEL calibration (Pejaver et al. 2022, Am J Hum Genet) (PMID:36413997)

Evidence considered

• Local REVEL predictor lookup: SF3B1 NM_012433.4:c.1874G>A (p.Arg625His), REVEL score 0.875.
• ClinGen SVI REVEL calibration (Pejaver et al. 2022, PMID:36413997): generic BP4 supporting threshold <= 0.29; the observed score does not meet it.
• SpliceAI max delta 0.049 was not used because this is a missense variant and the governing generic path requires REVEL only.
• Richards et al. 2015 ACMG/AMP guidance (PMID:25741868), Computational (In Silico) Predictive Programs: different in-silico tools are treated as a single piece of evidence rather than independent evidence; no double-counting was applied.

Sources

Reasoning

ClinVar contains an exact-match Likely pathogenic classification from a single laboratory, not a benign or likely benign expert-panel classification. Ordinary laboratory submissions, aggregate labels, and non-expert assertions do not satisfy the required BP6 trigger; therefore BP6 is not met.

Rule

Reputable source recently reports variant as benign, but the evidence is not available to the laboratory to perform an independent evaluation.

Evidence considered

• ClinVar variation 376534, exact variant NM_012433.4(SF3B1):c.1874G>A (p.Arg625His): Likely pathogenic, criteria provided, single submitter; zero expert-panel submissions.
• The available ClinVar record provides no benign or likely benign expert-panel assertion.

Still missing

• No exact-variant ClinVar expert-panel Benign or Likely benign classification is available.

Sources

Reasoning

The exact protein change SF3B1 p.Arg625His is reported in PMID:23861464, but the available source does not establish a previously known pathogenic variant suitable for the PS1 comparator requirement; the ClinVar record is a single-submitter somatic likely-pathogenic record and cannot alone establish the required pathogenic comparator.

Rule

Same amino acid change as a previously established pathogenic variant regardless of nucleotide change.

Evidence considered

• PMID:23861464 reports SF3B1 p.R625H in tumor 12 and in 8 additional uveal melanoma tumors, but does not establish the variant as a previously known pathogenic germline comparator.
• ClinVar variation 376534 contains one single-submitter somatic likely-pathogenic record; this is not an expert-panel or independently corroborated pathogenic classification.

Still missing

• An independently established pathogenic p.Arg625His variant record, with a reliable clinical or expert-panel classification, is needed.

Sources

Reasoning

No proband-level germline case report or parental testing data establish that SF3B1 c.1874G>A is de novo, and no family-history information is available. The ClinVar record is a somatic submission and does not provide maternity, paternity, or family evidence; therefore PS2 cannot be assessed.

Rule

De novo (both maternity and paternity confirmed) in a patient with the disease and no family history.

Evidence considered

• ClinVar variation 376534 records one exact-match submission as somatic, with no proband phenotype, parental genotypes, maternity confirmation, paternity confirmation, or family-history information.

Still missing

• A documented affected proband with the variant and the relevant disease phenotype.
• Confirmed maternity and paternity testing for the proband.
• Documented absence of family history for the relevant disease.

Sources

Reasoning

PMID:23861464 directly reports SF3B1 p.R625H (the queried c.1874G>A variant) in one discovery tumor and eight additional uveal melanoma tumors, and reports altered alternative-splicing associations for the SF3B1-mutated group. However, the extracted evidence does not establish a variant-specific functional result for p.R625H, does not provide independent replication or assay-control details sufficient to demonstrate a robust and reproducible assay, and does not show validation in a clinical diagnostic laboratory. Therefore the available evidence suggests a damaging biological association but is insufficient to apply PS3 or calibrate its strength under the required well-established functional-study definition.

Rule

Well-established in vitro or in vivo functional studies supportive of a damaging effect on the gene or gene product.

Evidence considered

• Liu et al. 2013, PMID:23861464: whole-genome sequencing of 12 primary uveal melanomas identified SF3B1 p.R625H in tumor 12; screening of 105 additional archival tumors identified 8 additional p.R625H tumors.
• The paper reports transcript and alternative-splicing analyses for SF3B1-mutated versus wild-type tumors, but the extracted finding does not isolate a p.R625H-specific functional effect or provide assay validation, controls, or independent replication adequate for a well-established PS3 assertion.

Still missing

• Variant-specific functional results for p.R625H with appropriate positive, negative, and wild-type controls.
• Independent replication or validated clinical-laboratory reproducibility sufficient for PS3 strength calibration.

Sources

Reasoning

The relevant full-text paper reports SF3B1 p.R625H in 1 of 12 discovery uveal melanoma tumors and 8 of 105 additional tumors, but it does not provide unaffected or population control counts for this exact variant and the observations are somatic tumor findings. Therefore, a case-control OR/RR and 95% CI cannot be calculated, so the PS4 requirement cannot be assessed. The governing rule requires a significantly increased prevalence in affected individuals versus controls and, under the supplied generic threshold, an OR/RR >5.0 with a confidence interval excluding 1.0.

Rule

The prevalence of the variant in affected individuals is significantly increased compared to the prevalence in controls. ... Note 1: Relative risk (RR) or odds ratio (OR), as obtained from case-control studies, is >5.0 and the confidence interval around the estimate of RR or OR does not include 1.0.

Evidence considered

• Ding et al. 2013, full text, Results paragraph beginning "Critically, we also observed non-recurrent mutations in SF3B1": tumor #12 carried C>T p.R625H; screening of 105 additional consecutive archival primary uveal melanomas detected 8 additional p.R625H tumors.
• The paper reports affected tumor observations only; it does not report unaffected or population control counts for p.R625H, so no case-control OR/RR or 95% CI was computed.
• The observations are somatic uveal melanoma tumor findings and are not a germline case-control comparison.

Still missing

• Exact-variant unaffected or matched-ancestry population control counts are unavailable.
• A case-control OR or RR and 95% confidence interval cannot be calculated from the available report.

Sources

Reasoning

PM3 cannot be assessed because no recessive SF3B1 disorder context, affected-proband observation, pathogenic second allele, or parent/offspring phase testing is documented. The available variant-specific report describes SF3B1 p.R625H as a somatic tumor mutation and does not establish detection in trans with a pathogenic variant. Thus, the required allelic configuration and inheritance context are unresolved.

Rule

For recessive disorders, detected in trans with a pathogenic variant ... This requires testing of parents (or offspring) to determine phase.

Evidence considered

• PMID:23861464 reports SF3B1 p.R625H in somatic uveal-melanoma tumors, including tumor 12 and 8 additional tumors, but provides no affected-proband germline observation, pathogenic second allele, or cis/trans phase information.
• ClinVar variation 376534 records the exact variant as a single-submitter somatic Likely pathogenic submission; this is not evidence of recessive inheritance or trans phase.
• The governing PM3 definition requires a recessive disorder and detection in trans with a pathogenic variant, with phase determined by testing parents or offspring.

Still missing

• A defined recessive SF3B1 disorder context is not documented.
• No pathogenic second variant and no parent/offspring testing or other validated phase information are documented.

Sources

Reasoning

PMID:23861464 reports alternate missense substitutions at R625 (p.Arg625Cys, p.Arg625Pro, and p.Arg625Leu), but the available extract does not determine any of them to be pathogenic. The Cancer Hotspots and COSMIC records establish recurrence or somatic occurrence, not the pathogenic comparator required by PM5.

Rule

Novel missense change at an amino acid residue where a different missense change determined to be pathogenic has been seen before.

Evidence considered

• PMID:23861464 reports alternate R625 substitutions p.R625C, p.R625P, and p.R625L, but does not provide pathogenic classifications for them.
• Cancer Hotspots and COSMIC document hotspot or somatic recurrence, which does not by itself establish a different R625 missense variant as pathogenic.

Still missing

• A different missense variant at residue 625 with an independently established pathogenic classification is needed.

Sources

Reasoning

No proband-level germline case report establishes assumed de novo occurrence of SF3B1 c.1874G>A, and there is no information about whether either parent was tested. The available ClinVar record is a somatic submission without parental or family data; therefore PM6 cannot be assessed.

Rule

Assumed de novo, but without confirmation of paternity and maternity.

Evidence considered

• ClinVar variation 376534 records one exact-match submission as somatic and provides no parental genotypes, parental testing status, proband phenotype, or family-history information.

Still missing

• A documented affected proband with the variant and the relevant disease phenotype.
• Evidence that the variant is absent from tested parent(s), with the extent of maternity and paternity confirmation documented.

Sources

Reasoning

No affected family members, informative meioses, segregation results, or phenotype concordance data are provided for SF3B1 c.1874G>A. The available ClinVar record is a somatic submission and contains no family segregation observations; therefore PP1 cannot be assessed or upgraded.

Rule

Co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease.

Evidence considered

• ClinVar variation 376534 records one exact-match somatic submission and provides no pedigree, affected-relative genotypes, phenotype data, or segregation analysis.

Still missing

• A family pedigree identifying affected relatives.
• Variant genotypes and disease phenotypes for affected and informative unaffected relatives.
• A disease relationship definitively attributable to SF3B1 for the segregating family.

Sources

Reasoning

The available evidence shows recurrent SF3B1 missense mutations in cancer, including p.Arg625His, but does not establish both required gene-level elements: a low rate of benign missense variation and missense variants as a common disease mechanism under a defined generic threshold.

Rule

Missense variant in a gene that has a low rate of benign missense variation and where missense variants are a common mechanism of disease.

Evidence considered

• PMID:23861464 documents recurrent SF3B1 missense mutations in uveal melanoma, including p.R625H, but does not quantify benign missense variation across SF3B1 or establish the complete PP2 premise.
• The queried allele is rare in gnomAD v4.1, but single-variant rarity is not a gene-level benign-missense constraint assessment.

Still missing

• A validated gene-level assessment of benign missense constraint and disease-causing missense mechanism is required.

Sources

Reasoning

No patient phenotype or family-history information for the queried case is provided in the available evidence. The available paper concerns somatic uveal melanoma tumor cohorts and does not establish that this patient's phenotype is highly specific for a disease with a single genetic etiology. PP4 therefore cannot be assessed.

Rule

Patient’s phenotype or family history is highly specific for a disease with a single genetic etiology.

Evidence considered

• No case-level phenotype or family-history description is present in the supplied evidence.
• PMID:23861464 reports somatic uveal melanoma tumor cohorts, not a case-level phenotype assessment for this variant.

Still missing

• Patient phenotype and family history are unavailable.
• A disease context with a sufficiently specific single genetic etiology is not documented.

Sources

Reasoning

BA1 requires a qualifying population frequency above 0.05. gnomAD v4.1 reports total AF 6.195740799944486e-07 (1/1614012 alleles), far below 0.05, but the required filtering frequency (grpmax FAF) is null in the available gnomAD evidence. Because BA1 must be adjudicated using grpmax FAF rather than a popmax point estimate, the criterion cannot be formally assessed from the available filtering metric.

Rule

BA1 (stand-alone): >= 0.05 — ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868)

Evidence considered

• gnomAD v4.1 total frequency: AF=6.195740799944486e-07, 1/1614012 alleles, homozygotes=0.
• The gnomAD record reports grpmax FAF=null; per the governing population-frequency instruction, BA1 must use grpmax FAF and not the popmax point estimate.
• Published generic threshold supplied for this case: BA1 >=0.05, ACMG/AMP 2015 stand-alone benign allele frequency (PMID:25741868).

Still missing

• gnomAD grpmax FAF is not available for the variant, so the specified filtering-frequency metric cannot be evaluated directly.

Sources

Reasoning

BS1 requires an allele frequency greater than expected for the disorder and must use grpmax FAF, not a popmax point estimate. gnomAD v4.1 reports total AF 6.195740799944486e-07 (1/1614012 alleles), far below the generic 0.01 default, but grpmax FAF is null. The available data therefore do not provide the required filtering-frequency value for formal BS1 adjudication; no disease-specific maximum credible allele-frequency calculation is available in the case evidence.

Rule

Allele frequency is greater than expected for the disorder.

Evidence considered

• gnomAD v4.1 total frequency: AF=6.195740799944486e-07, 1/1614012 alleles, homozygotes=0.
• The gnomAD record reports grpmax FAF=null; the specified BS1 method requires grpmax FAF rather than the popmax point estimate.
• Generic BS1 definition: allele frequency is greater than expected for the disorder; the supplied generic default is >=0.01, with per-gene derivation preferred under Whiffin et al. 2017 (PMID:28518168).

Still missing

• gnomAD grpmax FAF is unavailable.
• No prevalence, penetrance, allelic-heterogeneity, or maximum-credible-frequency inputs are available for a disease-specific BS1 calculation.

Sources

Reasoning

BS2 requires observation in a healthy adult individual, with the relevant zygosity for the inheritance model and full penetrance expected at an early age. The population evidence reports no homozygotes and one total heterozygous allele, but supplies no individual-level health status, age, phenotype, or inheritance-model context. Aggregate gnomAD allele counts cannot establish BS2.

Rule

Observed in a healthy adult individual for a recessive (homozygous), dominant (heterozygous), or X-linked (hemizygous) disorder, with full penetrance expected at an early age.

Evidence considered

• gnomAD v4.1 reports total AC=1, AN=1614012, and homozygotes=0; these aggregate counts do not identify a healthy adult individual or establish penetrance.

Still missing

• No individual-level carrier record with healthy-adult status, age, phenotype, zygosity, and relevant inheritance model is available.

Sources

Reasoning

The reviewed evidence reports SF3B1 p.R625H in uveal melanoma tumors and associates SF3B1-mutated tumors with alternative-splicing changes; it does not provide a well-established in vitro or in vivo assay showing that p.R625H has no damaging effect on protein function or splicing. No benign functional result with suitable controls and validated reproducibility was identified, so BS3 cannot be applied.

Rule

Well-established in vitro or in vivo functional studies show no damaging effect on protein function or splicing.

Evidence considered

• PMID:23861464 reports tumor-associated p.R625H and altered splicing associations for the SF3B1-mutated group, not a benign functional result for p.R625H.

Still missing

• A well-established benign functional assay for p.R625H assessing protein function or splicing, with appropriate controls and reproducibility.

Sources

Reasoning

No affected family members with negative testing for SF3B1 c.1874G>A are documented, and no pedigree or phenotype data permit evaluation of non-segregation. The available ClinVar record is a somatic submission without family observations; therefore BS4 cannot be assessed.

Rule

Lack of segregation in affected members of a family.

Evidence considered

• ClinVar variation 376534 records one exact-match somatic submission and provides no affected-relative testing, pedigree, phenotype, or non-segregation data.

Still missing

• A documented family with affected members evaluated for the variant.
• Reliable genotype results showing the variant is absent in affected members.
• Phenotype and family-structure information sufficient to address phenocopy and multiple-pathogenic-variant caveats.

Sources

Reasoning

The available evidence is insufficient to establish that SF3B1 disease is caused primarily by truncating variants. PMID:23861464 instead documents recurrent disease-associated somatic missense substitutions, but that cancer-focused observation cannot by itself resolve the gene-wide BP1 mechanism requirement.

Rule

Missense variant in a gene for which primarily truncating variants are known to cause disease.

Evidence considered

• PMID:23861464 reports recurrent SF3B1 missense substitutions in uveal melanoma, but it is not a comprehensive gene-level analysis of truncating versus missense disease mechanisms.

Still missing

• A gene-level disease-variant review comparing truncating and missense mechanisms is needed to assess BP1.

Sources

Reasoning

BP2 cannot be assessed because no affected-proband or familial observation documents this variant with a pathogenic allele, and no validated testing establishes either trans phase in a fully penetrant dominant disorder or cis phase in any inheritance pattern. The available variant-specific report is somatic and does not provide the required allelic relationship.

Rule

Observed in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observed in cis with a pathogenic variant in any inheritance pattern.

Evidence considered

• PMID:23861464 reports the exact p.R625H variant in somatic uveal-melanoma tumors but does not report a pathogenic partner variant or cis/trans phase.
• ClinVar variation 376534 identifies a single-submitter somatic Likely pathogenic record, without paired-allele or phase data.
• The governing BP2 definition requires observation in trans with a pathogenic variant for a fully penetrant dominant gene/disorder or observation in cis with a pathogenic variant in any inheritance pattern.

Still missing

• No pathogenic partner variant is documented in the same individual or tumor sample.
• No parent, offspring, or molecular phasing data establish cis or trans configuration.

Sources

Reasoning

The available evidence does not document a case-level alternate molecular basis for disease in an affected individual carrying this variant. The somatic tumor observations and the ClinVar submission do not establish such an alternate molecular diagnosis. BP5 therefore cannot be assessed.

Rule

Variant found in a case with an alternate molecular basis for disease.

Evidence considered

• No case report or clinical record in the supplied evidence documents an affected carrier with an alternate molecular basis for disease.
• PMID:23861464 reports tumor sequencing observations but does not establish the BP5 case scenario.

Still missing

• Case-level clinical information is unavailable.
• No alternate molecular diagnosis is documented for an affected carrier.

Sources